PHAGE-ATAC: a novel approach for rapid nanobody generation

Many sequencing technologies have been developed to characterize the cell state including RNA-seq, ATAC-seq, and ChIP-seq. It has become highly desirable to apply multiple techniques to the same cell. PHAGE-ATAC runs single cell ATAC-seq and mitochondrial DNA sequencing while also detecting intracellular epitopes. One key application of PHAGE-ATAC is the rapid generation of antigen specific nanobodies in weeks. In this post, we will cover the technologies incorporated in PHAGE-ATAC and explore how it works.

What are Phage Display and ATAC-Seq?

Phage display is commonly used in the directed evolution of enzymes and in vitro selection of antibodies, which has led to FDA-approved antibodies. Phage display uses a bacteriophage to tie a protein to a plasmid containing its DNA sequence. This allows for researchers to “sequence” a protein.

A major thrust of research has been studying organization of DNA in the nucleus. In general, there are two states of chromatin: open and closed. In closed chromatin, tightly packed nucleosomes block transcription, while in the open state DNA is actively transcribed. The open chromatin is positively correlated with gene expression and can be used to define cell fate. ATAC-seq is the current gold standard in methods of determining chromatin accessibility [1]. It works by using a transposase that transfers an oligo preferentially into open DNA. Amplification via primers targeting the oligo and sequencing identifies accessible chromatin.

How does PHAGE-ATAC work?

Multiomics is the application of different high throughput -omic assays to the same sample. Single cell RNA-seq cannot identify a cell’s surface markers well so techniques such as CITE-seq have been developed to tag the surface protein with an antibody linked to an oligo barcode [2]. This has proven very useful in scRNA-seq of immune cells as CD4+ and CD8+ populations can be reliably identified. This approach quantifies proteins and mRNA at the single cell level and can be alternatively used with scATAC-seq in ASAP-seq [3]. However, the oligo-barcoded antibodies are limited and not scalable.

Fiskin et al. developed PHAGE-ATAC, a process that uses nanobody displaying phages to recognize surface or intracellular epitopes [4]. There are three primary components: the nanobody, the linker containing a PHAGE-ATAC tag, and the p3 coat protein (Fig. 1). The phagemid encodes these three components and can be sequenced using next-generation sequencing. Bioinformatics pipelines are then used to analyze and determine which phagemid nanobodies were bound to the surface of each cell.

Figure 1: PHAGE-ATAC design (a) structure and sequence of PHAGE-ATAC phagemid along with (b) sequencing tag for amplification. (c) Experimental flowthrough of PHAGE-ATAC using 10X Genomics scATAC-seq [4]. 

This technique is capable of simultaneously indexing many phagemids and ATAC fragments in the same library prep. PHAGE-ATAC allows for detection of intracellular epitopes while also sequencing mitochondrial DNA. The authors showed that it can be used for cell hashing so that users can multiplex samples, superload to reduce batch effects, increase the amount of cells profiled, and identify doublets/multiplets.

PHAGE-ATAC Nanobody Library (PANL)

PHAGE-ATAC can rapidly generate antigen specific nanobodies in weeks. In the study, the researchers started with a synthetic high complexity library containing billions of phagemids [4]. The nanobodies were enriched through multiple rounds of selection and were also counter selected against parental cells that do not express the epitope. In an application of this library to identify a nanobody targeting enhanced Green Fluorescent Protein (eGFP), the top clones were shown to have similar amino acid sequences and 95% showed strong binding to eGFP (Fig. 2).

Figure 2: Rapid screening of nanobodies targeting surface eGFP [4]

Since recent SARS-CoV-2 variants have been able to spread much faster, there is a need for the development of rapid antigen testing. To see if PHAGE-ATAC could be used for detecting these epitopes at the host level, the authors screened 28 published nanobodies against the SARS-CoV-2 spike protein and selected the top seven for a panel that also contained eGFP nanobodies and immune cell surface marker nanobodies. In a dataset of nearly 5000 cells, they were able to differentiate between cells expressing eGFP, cells expressing SARS-CoV-2 spike protein, and immune cells [4]. This shows that PHAGE-ATAC is also able to detect viral and host epitopes at the single cell level.

Overall, PHAGE-ATAC is a significant advance in multiomics through the combination of single cell ATAC-seq and phage display. The application of this approach to the generation of nanobodies shows the clinically translational potential of this technique.

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  1. Buenrostro, J. D., Wu, B., Chang, H. Y., & Greenleaf, W. J. (2015). ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Current protocols in molecular biology109, 21.29.1–21.29.9.
  2. Stoeckius, M., Hafemeister, C., Stephenson, W., Houck-Loomis, B., Chattopadhyay, P. K., Swerdlow, H., Satija, R., & Smibert, P. (2017). Simultaneous epitope and transcriptome measurement in single cells. Nature methods14(9), 865–868.
  3. Mimitou, E. P., Lareau, C. A., Chen, K. Y., Zorzetto-Fernandes, A. L., Hao, Y., Takeshima, Y., Luo, W., Huang, T. S., Yeung, B. Z., Papalexi, E., Thakore, P. I., Kibayashi, T., Wing, J. B., Hata, M., Satija, R., Nazor, K. L., Sakaguchi, S., Ludwig, L. S., Sankaran, V. G., Regev, A., … Smibert, P. (2021). Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells. Nature biotechnology39(10), 1246–1258.
  4. Fiskin, E., Lareau, C. A., Ludwig, L. S., Eraslan, G., Liu, F., Ring, A. M., Xavier, R. J., & Regev, A. (2021). Single-cell profiling of proteins and chromatin accessibility using PHAGE-ATAC. Nature biotechnology40(3), 374–381.